Characterization of Plant-Fungal Interactions Involving Necrotrophic Effector-Producing Plant Pathogens

Abstract

Recently, great strides have been made in the area of host-pathogen interactions involving necrotrophic fungi. In this article we describe a method to identify, produce, and characterize effectors that are important in host-necrotrophic fungal pathogen interactions, and to genetically characterize the interactions. The main strength of this method is the combined use of pathogen inoculation, a pathogen culture filtrate bioassay, and genetic analysis of susceptibility and sensitivity in segregating host-mapping populations. These methods have been successfully used to identify several Stagonospora nodorum necrotrophic effectors and to characterize the genetic and phenotypic effects of individual host-effector interactions in the wheat-S. nodorum system. S. nodorum isolates that induce a differential response on two lines are used to produce culture filtrates that contain necrotrophic effectors while the wheat lines differing in reaction to the pathogen are used to develop a mapping population. The wheat population is used to develop DNA marker-based genetic linkage maps and culture filtrates are infiltrated across the mapping population. Linkage and quantitative trait loci (QTL) analysis is used to identify regions of the wheat genome harboring genes that govern sensitivity to necrotrophic effectors. The same populations are inoculated with the effector-producing isolate to determine the significance and proportion of disease explained by individual host gene-effector interactions. Additionally, from this information, differential lines that are sensitive to single effectors are developed for further purification and characterization of the effectors, eventually resulting in the identification, molecular cloning, and characterization of the effector genes.

Local adaptation drives the diversification of effectors in the fungal wheat pathogen Parastagonospora nodorum in the United States

“…These isolates were chosen for their diversity in geographical origin of isolation, as well as the differences in wheat class (spring, winter, and durum wheat). P. nodorum isolates were cultured as described by Friesen and Faris [69]. Briefly, dried agarose plugs stored at −20 °C of each isolate were brought to room temperature and rehydrated by placing on V8-potato dextrose agar (150 mL V8 juice, 3 g CaCO 3 , 10 g Difco PDA, 10 g agar, 850 mL H 2 O).…”

“…Plants were grown in the greenhouse for approximately two weeks, until the two to three-leaf stage. Inoculations were conducted as described by Friesen and Faris [69]. Briefly, plants were inoculated using a paint sprayer until leaves were fully covered, until runoff.…”

The Wheat Snn7 Gene Confers Susceptibility on Recognition of the Parastagonospora nodorum Necrotrophic Effector SnTox7

“…The P. nodorum isolate Sn6 (Friesen et al, 2007) was used for the production of NE‐containing cultures and inoculum for disease evaluations. Culture filtrates were produced and assayed as described in Friesen and Faris (2012). For infiltrations, plants were grown in the greenhouse at a temperature of 20 to 25°C and a 16‐h photoperiod.…”

Genetic mapping using a wheat multi-founder population reveals a locus on chromosome 2A controlling resistance to both leaf and glume blotch caused by the necrotrophic fungal pathogen Parastagonospora nodorum

“…Three to four seeds of each MAGIC line were sown in plastic cones following the protocol listed above for the inoculation experiments. P. nodorum isolates were cultivated in liquid Fries 3 medium (Friesen and Faris 2012) for the production of necrotrophic effectors. Three weeks after the stationary phase, culture filtrates were sterilized filtered through membranes filters (white gridded: 0.45 μm, diameter: 47 mm, S-PAK, France) and roughly 50 μL culture filtrates or ToxA preparation were infiltrated into the second leaf of each plant by using a 1-mL syringe with the needle removed.…”